For the automation of flow cytometry-based assays of immune function, funding is requested to purchase a Beckman Coulter BioMek 2000 system, to be located in the Gladstone/UCSF Core Immunology Laboratory at San Francisco General Hospital. In the Core Laboratory. a flow cytometry-based assay system has been developed and optimized to detect specific CD4+ and CD8+ T cell responses to HIV-1 and to cytomegalovirus (CMV) in cohorts of HIV-1-infected patients. Unlike the MHC Class I tetramer assay, this ?cytokine flow cytometry (CFC) assay can be used to detect T cell responses against undefined antigenic epitopes in the context of uncharacterized MHC backgrounds. Related assays are being developed for the assessment of specific human CD4+ and CD8+ T cell responses to other AIDS-related opportunistic infections, including those caused by Mvcobacterium tuberculosis, the Mvcobacterium avium complex, Pneumocvstis carinii, and human papilloma virus. Many investigators at UCSF have incorporated CFC into their research protocols; consequently the number of CFC assays performed in the Core Laboratory is steadily and rapidly increasing. To keep up with the demand for this service, to improve safety and reliability, and to perform experiments that would be impractical or impossible to perform manually, it is necessary to automate CFC. Combined with the instrumentation already in place, the proposed instrument system will allow for the automation and further development of CFC assays, significantly strengthening and enhancing the ability of the Core Immunology Laboratory to support the many Gladstone/UCSF investigators engaged in HIV research. In addition, these automated assays will be made available to scientists engaged in other areas of research and to scientists from other parts of the country (e.g., through the AIDS Clinical Trials Group). The scientific interests and expertise of the selected major users include a broad spectrum of biology and a unifying focus on the analysis of immune function; consequently, the instrument system will support a broad range of projects involving the evaluation of T cell responses in the context of: 1) HIV-1 vaccines, 2) antiretroviral therapy failure, 3) structured treatment interruption, 4) cross clade recognition, 5) CTL dynamics, 6) HPV in cervical dysplasia, 7) Pneumocystis carinii infection, 8) solid organ transplantation, 9) "alternative therapy" with DHEA, and 10) CSF pleocytosis. In summary, this proposed instrument system is pivotal to the success of several NIH-funded investigations and researchers, will provide a critical resource to the Gladstone/UCSF research community, and will spark innovative research in the future.